Biochemical Analysis of Histone Succinylation

Figure 1

In vitro acylation assay using fractionated HepG2 cell extracts. (a) Schematic diagram of the fractionation of HepG2 cell extracts and in vitro acylation assay. (b) Validation of HepG2 cells fractionated into cytosolic, nuclear, and chromatin extracts. 4 μg of each extract was assessed by western blot (left panel). IKKα, HDAC2, and AcH3 were used as cytosolic, nuclear, and chromatin fraction markers, respectively. CBB staining of loading proteins is shown in the right panel. Cyt: cytosolic extracts; NEs: nuclear extracts; Chr: chromatin extracts. (c) In vitro acylation assays using fractionated cell extracts. ¹⁴C-labeled coenzyme A incorporation was assessed by liquid scintillation counting. Reaction mixtures without cell extracts were used as negative controls (mock). To show that the incorporation was an enzymatic reaction, cell extracts were heat-inactivated at 96°C for 10 min. Ac-CoA: acetyl-coenzyme A; Mal-CoA: malonyl-coenzyme A; Suc-CoA: succinyl-coenzyme A.

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